The human genetic variation in Italy is the result of ancient population movements, demographic changes and geography. The increasing possibility of studying the genetic structure of selected Italian population samples at a high level of phylogenetic resolution provides a particularly useful model to assess the presence of genetic traces of the ancient people who lived in Italy in pre-Roman times in present populations. In this study we reconstructed the paternal and maternal genetic history of seven small villages, mountain communities in Central Italy, and identified possible genetic barriers between them. The involved communities, because of their geographic location, have experienced during centuries an evident geographic isolation and biodemographic stability. For investigating both the maternal and paternal genetic background, we analyzed 406 individuals for mtDNA, in particular D-Loop HVS I and HVS II and informative SNPs within the coding region, and 253 for Y chromosome STRs. All the mtDNA and Y chromosome haplogroups found are mainly of Western Eurasian origin, but there are some typical of Near East. The distribution of both mtDNA and Y chromosome diversity, although similar to that found in other European populations, shows the typical signs of a certain degree of isolation. The results suggest that, despite their close geographic proximity, each village has a typical genetic background due to a low degree of gene flow with the neighboring communities, probably related not only to the mountain features of this area, but also to a cultural separation.